The African trypanosome Trypanosoma brucei is the causative agent of sleeping sickness in humans and of related diseases in livestock. It lives in the bloodstream and survives by undergoing antigenic variation of its major Variant cell-Surface Glycoprotein (VSG) coat. T. brucei depends on its host for nutrients for growth and development. The observation that macromolecules, such as low density lipoprotein (LDL) particles and transferrin enter trypanosomes via receptor-mediated endocytosis at the flagellar pocket, encouraged us to identify evolutionarily conserved surface proteins located in the flagellar pocket. The flagellar pocket of trypanosomes, representing about 0.43% of the pellicle membrane, is a deep invagination of the plasma membrane where the flagellum extends from the cell. In this pocket, the integrity of the VSG coat is broken and the subpellicular microtubule sheath is absent. Thus, the flagellar pocket forms a structurally and functionally discrete surface domain of Trypanosomatids and has been considered to be the only site, where receptor mediated endocytosis takes place in trypanosomes. The identification of invariant surface protein localized in the flagellar pocket and studies of their functional properties may have practical applications for parasite control, and will give us valuable insights into the mechanisms involved in endocytosis of macromolecules and the biology of host-parasite interactions. We have identified several genes encoding cell-surface proteins located at the flagellar pocket of T. brucei. The genes for these proteins are conserved in diverged species of Kinetoplastida (T. brucei, T. equiperdum, T. lewisi and T. cruzi) indicating their potential functional significance. One of the identified proteins is a Cysteine-Rich Acidic- repetitive transMembrane (CRAM) protein located in the flagellar pocket of T. brucei. Our recent study provided evidence for CRAM serving as a lipoprotein receptor in the procyclic form of T. brucei. Additional studies identified a putative retention signal which functions to confine CRAM to the flagellar pocket. In this proposal, we will focus on: 1) studying the mechanism of retention of proteins in the flagellar pocket; 2) determining whether a similar sorting system is present in both stages of T. brucei; and 3) determining the role of the cytoplasmic extension of CRAM in the process of internalization.